BIOTECHNOLOGY LETTERS Volume I7 No. 7 (July 1995) p/1757- 760 Received 26th May

INCREASED ETHANOL PRODUCTION DURING GROWTH OF ELECTRIC-FIELD STIMULATED KLUWEROMYCES MA~US IMB3 DURING GROWTH ON LACTOSE-CONTAINING MEDIA AT 45OC.

J. Simpson, D. Brady, A. Rollan, N. Barron, L.McHale & A.P.McHale.

Biotechnology Research Group, School of Applied Biological and Chemical Sciences, University of Ulster, Coleraine, Co. Londondeny, BT52 lSA, Northern Ireland.

SUMMARY

Following treatment of the thermotolerant ethanol producing yeast strain Kluyveromyces marxianus IMB3 with single electric field pulses of 2.4kV/cm, ethanol production was found to increase from 25 to 35% of the theoretical maximum yield within 20 hours, during growth on lactose-containing media (4% [w/v]). Following treatment with electric fields and addition of 1mM MnCl, to lactose-containing media, ethanol production almost doubled within a 30 hour period. In addition, low levels of functional P-galactosidase activity could be detected in the extracellular culture filtrates, presumably as a result of electropermeabilization.

INTRODUCTION

In recent studies it was demonstrated that the thermotolerant ethanol-producing yeast, Kluyveromyces marxianus IMB3 is capable of ethanol production during growth on lactose containing media at 45°C (Brady et al., 1994). The commercial implication of the latier, particularly with respect to treatment of dairy industry residues such as whey and whey permeates is well recognized (Mawson, 1987; Vienne & von Stockar, 1985).

In our studies relating to ethanol production during growth of the above mentioned organism on lactose-containing media at relatively elevated temperatures, it has been demonstrated that the organism produces a cell associated p-galactosidase (Brady et al., 1995). In addition it has been demonstrated that although the enzyme is relatively thermolabile at fermentation temperatures, addition of MnClz stabilizes the activity (Brady et a1.,1995a) and contributes to increased ethanol production.

In a recent report from our laboratory it has been shown that treatment of K. marxianus IMB3 cells with electric fields led to increased ethanol production following growth of the organism on cellobiose-containing media (McCabe et al., 1995). In that study it was suggested that such treatments induced cell electropermeabilization and increased access of substrate to p- glucosidase. In the study presented here it was decided to determine whether or not electropermeabilization of the cells using the previously reported technique. would contribute significantly to increased ethanol production by K. marxianzrs IMB3 during growth on lactose- containing media.

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The microorganism: The thermotolerant yeast strain K. marxianus IMB3 was maintained on malt extract agar slopes at 45°C. Submerged cultum in lactose-containing media was carried out in shake flasks at 45°C in an orbital incubator as described previously (Brady et al, 1994).

Electropermeabilization: Cells from 20ml fermentations cultured for 14 hrs. on lactose- containing medium (4% w/v) were harvested by centrifugation. The cell pellet was washed in electroporation medium consisting of 3g/L malt extract, 3g5 yeast extract and 5g/L peptone, pH 5.5. The pellet (approx. 0.2ml) was resuspended in 0.8ml electroporation medium and the total vol. was dispensed into electroporation cuvettes with an electrode gap of 0.4cm. Pulses were generated using a Bio-Rad Gene Pulser (Bio-Rad, Herts., UK) and conditions were arranged in order to ensure delivery of pulses with electric field strengths of 2,400 V/cm. Control samples were treated in a similar mamrer except that an electric pulse was not delivered. Following treatment, cell suspensions were dispensed into 19.2ml of yeast growth medium containing 4% (w/v) lactose (Brady et al., 1994). Fermentations were then carried out at 45°C as described previously (Brady et al., 1994).

p-galactosiaizse assay: Enzyme activity was determined using the substrate o-nitrophenyl+D- galactoside as described previously (Brady et al., 1994). Activity is expressed as nmoles of o- nitrophenol produced per min. per ml of culture filtrate.

Determination of ethanol concentration: Ethanol concentration was determined using a gas chromatograph (GLC 8410, Perkin Elmer) as described previously by Fleming et al., 1993).

RESULTS AND DISCUSSION

It has previously been demonstrated that the thermotolerant yeast K. marxianus IMB3 is capable of producing significant quantities of ethanol during growth on lactose-containing media (Brady et al., 1994). During those studies it was found that yields of ethanol were relatively low, even when media were supplemented with p-galactosidase activity. Since it was recently demonstrated that exposure of K marxianus IMB3 cells to electric field pulses increased ethanol production during growth on cellobiose (McCabe et al., 1995), it was of interest to determine whether or not similar treatments would contribute to increased ethanol production following growth of the treated cells on lactose-containing media. To this end cell populations were treated with electric field pulses as described in the Methods section and subsequently cultured in lactose-containing media. Control samples consisted of fermentations inoculated with untreated cells. Samples were harvested at the indicated times and ethanol production was examined. The results obtained are shown in Fig. 1A and they demonstrate that in control samples ethanol production reached approximately 5g/L within 2Ohrs., representing almost 25% of the theoretical maximum yield. It shouls be noted that hese results were in relatively close agreement with those previously reported (Brady et al., 1994). However, when cells which had been exposed to electric field stimulation were grown on lactose containing media ethanol production increased to 7.3g/L and this represented almost 35% of the maximum theoretical yield. This result demonstrates increased substrate utilization, although the increase in ethanol out put is not as impressive as that observed during our studies with the electric-field stimulated organism grown on cellobiose-containing media (McCabe et al., 1995).

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In our previous work it was shown that only trace amounts of extracellular p- galactosidase activity could be detected in the culture filtrates (Brady et al., 1994). It was also shown that when lactose-containing media were supplemented with the cell-associated p- galactosidase from K. marxiunus IMB3, that activity disappeared from the medium within a short period of time and this was shown to be the result of inactivation by the operating temperatures of the system.

f 3 d i E

7.s-

S-

2.5 -

0 k’ n 10 2n .w 4n sn

TIME (II)

Fig.lA

n In zn .3n 40 sn 60

TIMF. (h)

Fig.lB

Fig.lA Ethanol production by normal (0 - 0) and electric-field stimulated (0 - 0) K murxiunus IMB3 cells on lactose-containing media at 45°C. Fig.lB Ethanol production by electric field stimulated K murxiunus IMB3 during growth on lactose containing media in the presence (A - A ) and absence (0 - 0) of 1mM Mn2.‘. Controls (0 - 0 ) consisted of intact cells in the presence and absence of 1mM Mr?+.

In subsequent studies it was demonstrated that the addition of Mn” to lactose containing media had a stabilizing effect on the K. murxiunus IMB3 P-galactosidase (Brady et al., 1995a) and it was further demonstrated that ethanol production reached 91% of the maximum theoretical yield. These results suggested that addition of Mn” to the electric field stimulated cells might prove beneficial. To this end electric field stimulated cells were added to lactose-containing media in the presence and absence of Mn”. Control samples consisted of fermentations containing non-stimulated cells in the presence and absence of Mn”‘. The results obtained are shown in Fig. 1B. When untreated cells were cultured in the presence and absence of Mn”, no significant difference in ethanol production was observed. However, when Mn’+ was added to the treated cells, ethanol production increased from 38 to 75% of the maximum theoretical yield. Although, as mentioned above the maximum previously reported ethanol out put during growth of this thermotolerant yeast strain on lactose was found to be 91% of maximum theoretical yield, it was necessary to add p-galactosidase to the growth medium (Brady et al., 1995). In practical

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terms this requirement would contribute to increased running costs of the system and as such could prove prohibitive. In this context the authors believe that electric-field stimulation of the strain prior to inoculation would tend to offer more in practical terms since the treatment procedure is both simple from a technical point of view and is also extremely rapid.

In our previous work with electric-field stimulated K. marxianus IMB3 cells and subsequent growth on cellobiose-containing media, the mechanism by which stimulation increased ethanol out put was unclear (McCabe et al., 1995). Since there was no apparent increase in extracellular P-glucosidase it was suggested that the increases in ethanol production resulted from increased permeability and therefore increased access of substrate to cell-associated enzyme. In order to determine whether or not electric-field stimulation resulted in release of p-galactosidase into the extracellular environment it was decided to assay culture filtrates for this activity. As shown previously, this enzyme was found to exist in only trace amounts in the extracellular culture filtrate during growth of the organism on lactose-containing media (Brady et al., 1995). When culture filtrates from cells treated with electric fields were assayed for this activity it was found that activities of 1.5U/ml culture filtrate could be detected at 20 hrs. growth. No activity could be detected in the extracellular culture filtrates from untreated cells. Therefore the results presented here suggest that increased ethanol production by the treated cells, at least in the lactose-containing system, results from leakage of enzyme into the culture medium. The results, when taken together with previous reports, further suggest that the increase in ethanol production in the presence of Mn2’ results from stabilization of that activity as shown previously (Brady et al., 1995a).

As in our previous work the results presented here suggest that electric field stimulation may play an important role in bioconversion systems where substrate accessibility to the cellular conversion machinery may be restricted.

Acknowledgement: D.B. was in receipt of a postgraduate scholarship from the Dept. of Education, Northern Ireland.

REFERENCES: Brady, D., Marchant, R., McHale, L. 62 McHale, A.P. (1994) Biotechnol. Let&. 16, 737-740.

Brady, D., Marchant, R., McHale, L. & McHale, A.P. (1995) Biotechnol. Letts. 17, 233-236.

Brady, D., Marchant, R., McHale, L. & McHale, A.P. (1995a) Enz. Microb. Technol. In Press.

Fleming, M., Barron, N., McHale, L., Marchant, R. & McHale, A.P. (1993) Biotechnol. Letts. 15, 1195-l 198.

Mawson, A.J. (1987) Australian J Biotechnol. 1, 64-67.

McCabe, A., Barron, N., McHale, L. & McHale, A.P. (1995) Biotechnol. Tech. 9, 133-136.

Vienne, P. & von Scockar, U. (1985) Biotechnol. Letts. 7, 521-526.

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